Sirp alpha-antibody fusion proteins

ABSTRACT

SIRPabodies comprise an immunoglobulin variable region, which may specifically bind a tumor antigen, viral antigen, etc., fused to a sequence comprising a binding domain of SIRPα. The binding domain of SIRPα comprises at least the N-terminal Ig-like domain of SIRPα, and may further comprise additional SIRPα sequences. The SIRPabodies find use in therapeutic methods that benefit from the combined activity of blocking CD47 activity, and antibody targeting, e.g. in the treatment of cancer, etc. In some specific embodiments, the SIRPabody comprises anti-CD20 activity and a SIRP binding domain; anti-CD99 and a SIRP binding domain; or anti-TIM3 activity and a SIRP α binding domain.

CROSS REFERENCE

This application is a 371 application and claims the benefit of PCTApplication No. PCT/US2015/044304, filed Aug. 7, 2015, which claimsbenefit of U.S. Provisional Patent Application No. 62/035,273, filedAug. 8, 2014, which applications are incorporated herein by reference intheir entirety.

BACKGROUND OF THE INVENTION

Macrophages clear pathogens and damaged or aged cells from the bloodstream via phagocytosis. Cell-surface CD47 interacts with its receptoron macrophages, SIRPα, to inhibit phagocytosis of normal, healthy cells.CD47 is a broadly expressed transmembrane glycoprotein with a singleIg-like domain and five membrane spanning regions, which functions as acellular ligand for SIRPα with binding mediated through the NH₂-terminalV-like domain of SIRPα. SIRPα is expressed primarily on myeloid cells,including macrophages, granulocytes, myeloid dendritic cells (DCs), mastcells, and their precursors, including hematopoietic stem cells.

SIRPα inhibits the phagocytosis of host cells by macrophages, where theligation of SIRPα on macrophages by CD47 expressed on the host targetcell generates an inhibitory signal mediated by SHP-1 that negativelyregulates phagocytosis. SIRPα acts to detect signals provided by “self,”to negatively control innate immune effector function against thesecells.

In keeping with the role of CD47 to inhibit phagocytosis of normalcells, there is evidence that it is transiently upregulated onhematopoietic stem cells (HSCs) and progenitors just prior to and duringtheir migratory phase, and that the level of CD47 on these cellsdetermines the probability that they are engulfed in vivo.

The present invention provides immunoglobulin fusion proteins thatinhibit the interaction of CD47 with SIRP leading to phagocytosis as aresult of disrupting the negative regulatory signal.

SUMMARY OF THE INVENTION

Compositions and methods are provided relating to fusion proteins,herein termed SIRPabodies, that comprise an immunoglobulin variableregion, which may specifically bind a tumor antigen, viral antigen,etc., fused to a sequence comprising a binding domain of SIRPα. Thebinding domain of SIRPα comprises at least the N-terminal Ig-like domainof SIRPα, and may further comprise additional SIRPα sequences. TheSIRPabodies find use in therapeutic methods that benefit from thecombined activity of blocking CD47 activity, and antibody targeting,e.g. in the treatment of cancer, etc. In some specific embodiments, theSIRPabody comprises an anti-CD20 binding domain and a SIRPα bindingdomain. In other specific embodiments the SIRPabody comprises ananti-CD99 binding domain and a SIRPα binding domain. In other specificembodiments the SIRPabody comprises an anti-TIM3 binding domain and aSIRPα binding domain.

SIRPabody polypeptide molecules of the invention comprise two functionaldomains: an immunoglobulin variable region domain, and the N-terminalIg-like domain of SIRPα, for example as shown in SEQ ID NO:1 andvariants thereof, including without limitation allelic polymorphisms. Insome embodiments the SIRPabody comprises a first and a secondpolypeptide chain, which first polypeptide chain comprises (i) a firstdomain comprising a binding region of a light chain variable domain ofan immunoglobulin (V_(L)) specific for a first epitope; and a secondpolypeptide comprising (i) a first domain comprising a binding region ofa heavy chain variable region domain of an immunoglobulin (V_(H))specific for a first epitope; and (ii) a second domain comprising theN-terminal Ig-like domain of SIRPα. In some embodiments, the firstpolypeptide and the second polypeptide further comprise the respectiveheavy and light chain constant region domains of the immunoglobulin,i.e. C_(L) and C_(H). In some embodiments, the second polypeptidecomprises the N-terminal Ig-like domain of SIRPα fused to the aminoterminus of the V_(H) domain (N-terminal SIRPabody, or NH-SIRPabody). Inother embodiments the second polypeptide comprises the N-terminalIg-like domain of SIRPα fused to the amino terminus of the C_(H) domains(C-terminal SIRPabody, or CH-SIRPabody).

In an alternative embodiment, the SIRPabody comprises a first and asecond polypeptide chain, which first polypeptide chain comprises (i) afirst domain comprising a binding region of a light chain variabledomain of an immunoglobulin (V_(L)) specific for a first epitope; and(ii) a second domain comprising the N-terminal Ig-like domain of SIRPα;and a second polypeptide comprising (i) a first domain comprising abinding region of a heavy chain variable region domain of animmunoglobulin (V_(H)) specific for a first epitope. In someembodiments, the first polypeptide and the second polypeptide furthercomprise the respective heavy and light chain constant region domains ofthe immunoglobulin, i.e. C_(L) and C_(H). In some embodiments, the firstpolypeptide comprises the N-terminal Ig-like domain of SIRPα fused tothe amino terminus of the V_(L) domain (NL-SIRPabody). In otherembodiments the second polypeptide comprises the N-terminal Ig-likedomain of SIRPα fused to the amino terminus of the C_(L) domains(CL-SIRPabody).

The SIRPα domain and the V_(H) or C_(H) domains may be separated by ashort linker. The peptide linker may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in length, and isof sufficient length and amino acid composition to minimize sterichindrance between the binding domains. In some embodiments the linker isglycine and/or serine; exemplary is one or more subunits of SEQ ID NO:2.Alternatively the sequence of the constant region can provide thelinker, for example as shown in SEQ ID NO:3.

The SIRPabodies of the invention are particularly efficacious in thetreatment of disease, e.g. increasing phagocytosis of living CD47expressing cells. Treatment may be systemic or localized, e.g. deliveryby intratumoral injection, etc. In certain embodiments, methods areprovided for a targeted cytotoxicity therapy that (a) blocks CD47activity through the SIRPα binding domain and may additionally (b)provide antibody mediated targeting and/or cell killing as a result ofbinding to the immunoglobulin variable region domain. A targeted cellpopulation suspected of expressing on the cell surface the cognateantigen for the immunoglobulin variable region domain of a SIRPabody iscontacted with an effective dose of the SIRPabody, where the dose issufficient to increase killing of the targeted cells, relative to thelevel of killing obtained with the immunoglobulin alone. In suchembodiments, the F_(c) region of the immunoglobulin is generallypresent, and the activity may be compared to the activity of the intactimmunoglobulin.

In some such embodiments the cognate antigen is CD20. Antibodies thatspecifically target CD20 are well known in the art, including human andhumanized antibodies available for therapeutic purposes, e.g.Tositumumab, Rituximab, Ibritumomab Tiuxetan, Veltuzumab, AME-133v,Ofatumumab, R7159, etc. The targeted cells in such an embodiment areCD20 positive cancer cells. The contacting may be performed in vivo, andincludes the treatment of malignancies, including without limitation Bcell non-Hodgkin lymphomas, hairy cell leukemia, B-cell chroniclymphocytic leukemia, melanoma cancer stem cells, etc. In someembodiments, treatment of an individual with such a malignancy comprisesthe steps of administering an effective dose of a SIRPabody that causesincreased killing of CD20+ malignant cells, i.e. to increase by greaterthan about 20%, to increase by greater than about 30%, to increase bygreater than about 40%, to increase by greater than about 50%, toincrease by greater than about 75%, to increase by greater than about90%, to increase by greater than about 95%, to increase by greater thanabout 99% or more relative to treatment with the correspondingimmunoglobulin lacking the SIRPα binding domain. A synergistic responsemay be obtained, where, for example, the reduction in tumor cellpopulation with the SIRPabody is greater than the reduction obtainedwith one or both of soluble SIRPα binding domain and anti-CD20immunoglobulin. SIRPabodies for this purpose include without limitationCD20-2GL-SIRPα, CD20-4GL-SIRPα, and SIRPα-CD20 provided herein.

In other embodiments the cognate antigen is CD99. Antibodies specificfor CD99 and are known in the art and commercially available, e.g. 12E7,HCD99, F21, O13, etc. CD99 is expressed on a number of cancers,including without limitation Ewing's sarcoma tumors, thymic tumors,synovial sarcoma, haemangiopericytoma, and meningioma, small cell lungcancer, AML, diffuse large B-cell lymphoma (DLBCL), etc. In someembodiments, treatment of an individual with such a malignancy comprisesthe steps of administering an effective dose of a SIRPabody that causesincreased killing of CD99+ malignant cells, i.e. to increase by greaterthan about 20%, to increase by greater than about 30%, to increase bygreater than about 40%, to increase by greater than about 50%, toincrease by greater than about 75%, to increase by greater than about90%, to increase by greater than about 95%, to increase by greater thanabout 99% or more relative to treatment with the correspondingimmunoglobulin lacking the SIRPα binding domain. A synergistic responsemay be obtained, where, for example, the reduction in tumor cellpopulation with the SIRPabody is greater than the reduction obtainedwith one or both of soluble SIRPα binding domain and anti-CD99immunoglobulin.

In other embodiments the cognate antigen is TIM3. Antibodies specificfor TIM3 and are known in the art and commercially available, e.g.1F38-2E2, etc. CD99 is expressed on certain cancer cells, includingwithout limitation AML. In some embodiments, treatment of an individualwith such a malignancy comprises the steps of administering an effectivedose of a SIRPabody that causes increased killing of TIM3+ malignantcells, i.e. to increase by greater than about 20%, to increase bygreater than about 30%, to increase by greater than about 40%, toincrease by greater than about 50%, to increase by greater than about75%, to increase by greater than about 90%, to increase by greater thanabout 95%, to increase by greater than about 99% or more relative totreatment with the corresponding immunoglobulin lacking the SIRPαbinding domain. A synergistic response may be obtained, where, forexample, the reduction in tumor cell population with the SIRPabody isgreater than the reduction obtained with one or both of soluble SIRPαbinding domain and anti-TIM3 immunoglobulin.

In other related embodiments, an anti-CD20 SIRPabody is used in a methodof treating autoimmune diseases with a B cell component, includingwithout limitation rheumatoid arthritis, multiple sclerosis, Type Idiabetes, Type II diabetes, systemic lupus erythematosus, and the like.In such methods targeted cytotoxicity (a) blocks CD47 activity throughthe SIRPα binding domain and (b) provides antibody mediated cell killingthrough the immunoglobulin variable region domain. A targeted B cellpopulation expressing CD20 is contacted with an effective dose of theSIRPabody, where the dose is sufficient to increase killing of thetargeted cells, relative to the level of killing obtained with theimmunoglobulin alone. In such embodiments, the F_(c) region of theimmunoglobulin is generally present, and the activity may be compared tothe activity of the intact immunoglobulin.

In yet another embodiment of the present invention, the SIRPabodies ofthe invention can be used to treat a variety of diseases and disorders.Accordingly, the present invention is directed to a method for treatinga disease or disorder comprising administering to a patient in needthereof an effective amount of a SIRPabody of the invention, in whichthe SIRPabody has been selected to provide a specific level of targetingcorrelated with the binding specificity of the immunoglobulin, coupledwith blocking CD47 activity. Such methods include those contemplated byU.S. Pat. No. 8,562,997, international applications US2014/014905 andUS2014/035167, each herein specifically incorporated by reference. Infurther such embodiments of the invention, the antigen is a tumorantigen.

The immunoglobulin portion of the SIRPabody may comprise constant regionsequences that are characteristic of mouse, rabbit, primate, human,etc., antibodies. In some embodiments, antibody sequence elements arehumanized, primatized, chimeric, etc., as is known in the art. Moreover,the term “antibody” as used herein, can refer in appropriate embodiments(unless otherwise stated or clear from context) to any of the art-knownor developed constructs or formats for utilizing antibody structural andfunctional features in alternative presentation. For example,embodiments, an immunoglobulin portion of the SIRPabody utilized inaccordance with the present invention is in a format selected from, butnot limited to, IgG, IgE and IgM, bi- or multi-specific antibodies(e.g., Zybodies®, etc), single chain Fvs, polypeptide-Fc fusions, Fabs,cameloid antibodies, masked antibodies (e.g., Probodies®), Small ModularImmunoPharmaceuticals (“SMIPs™”), single chain or Tandem diabodies(TandAb®), VHHs, Anticalins®, Nanobodies®, minibodies, BiTE®s, ankyrinrepeat proteins or DARPINs®, Avimers®, a DART, a TCR-like antibody,Adnectins®, Affilins®, Trans-bodies®, Affibodies®, a TrimerX®,MicroProteins, Fynomers®, Centyrins®, and a KALBITOR®.

For example, the SIRPabody may comprise a full length chimeric orhumanized antibody, e.g. having a human immunoglobulin constant regionof any isotype, e.g. IgG1, IgG2a, IgG2b, IgG3, IgG4, IgA, etc. or anantibody fragment, e.g. a F(ab′)₂ fragment, and F(ab) fragment, etc.Furthermore, the SIRPabody may be labeled with a detectable label,immobilized on a solid phase and/or conjugated with a heterologouscompound.

The invention further provides: isolated nucleic acid encoding theSIRPabodies and variants thereof; a vector comprising that nucleic acid,optionally operably linked to control sequences recognized by a hostcell transformed with the vector; a host cell comprising that vector; aprocess for producing the antibody comprising culturing the host cell sothat the nucleic acid is expressed and, optionally, recovering theantibody from the host cell culture (e.g. from the host cell culturemedium). The invention also provides a composition comprising one ormore SIRPabodies and a pharmaceutically acceptable carrier or diluent.This composition for therapeutic use is sterile and may be lyophilized,e.g. being provided as a pre-pack in a unit dose with diluent anddelivery device, e.g. inhaler, syringe, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIG. 1A-1B: Schematic of SIRPα-rituximab reagents (SIRPabodies). TheN-terminal immunoglobulin fold from wild type SIRPα (green) wasengineered onto either the N or C terminus of the heavy chain ofrituximab (clone 2B8). The light chain sequence is provided as SEQ IDNO:4, where the signal sequence is amino acid residues 1-22. Apolyglycine serine linker SEQ ID NO:2, (GGGGS)₂ or (GGGGS)₄ was used tofuse SIRPα onto the C terminus of the heavy chain to createCD20-2GL-SIRPα (SEQ ID NO:6) or CD20-4GL-SIRPα (SEQ ID NO:10),respectively. The signal sequence is amino acid residues 1-19, thelinker is residues 471-480 or 471-490, respectively. The SIRPα sequenceis residues 481-599, or 490-609, respectively. A linker derived from theN terminal amino acids of the CH1 domain SEQ ID NO:3, (ASTKGPSVFPLAP)was used to fuse SIRPα onto the N terminus of the heavy chain, shown asSEQ ID NO:8, where the signal sequence is amino acid residues 1-17, theSIRPα sequence is residues 18-136, the linker is residues 137-149.

FIG. 2: Construction of SIRPα-rituximab bispecific reagents. SDS-PAGEanalysis of the indicated purified antibodies under non-reducing (left)and reducing (right) conditions. Anti-CD20 antibody was included as areference for the sizes of the parental heavy and light chains.

FIG. 3A-3B: SIRPα-rituximab bispecific reagents bind to CD20 on the cellsurface. The indicated antibodies were used to stain rat YB2/0 cellsengineered to express human CD20, but not human CD47 (CD20+CD47−) priorto detection with anti-human secondary antibody by flow cytometry.2B8=anti-CD20, B6H12=anti-CD47.

FIG. 4: SIRPα-rituximab bispecific reagents bind to CD20 with similaraffinity as parental antibody. CD20+CD47− YB2/0 cells were incubatedwith the indicated primary antibodies over a range of concentrationsprior to staining with 10 μg/ml DyLight 488anti-CD20 and detection byflow cytometry. Mean fluorescence intensity (MFI) of the DyLight 488signal for each condition was measured by flow cytometry.

FIG. 5A-5B: SIRPα-rituximab bispecific reagents bind to CD47 on the cellsurface with reduced affinity relative to anti-CD47. The indicatedantibodies were used to stain rat YB2/0 cells engineered to expresshuman CD47, but not human CD20 (CD20−CD47+) prior to detection withfluorescently tagged anti-human secondary antibody by flow cytometry.2B8=anti-CD20, B6H12=anti-CD47.

FIG. 6: Binding of SIRPα-rituximab bispecific reagents to CD47 isoutcompeted by labeled anti-CD47. CD20−CD47+ YB2/0 cells were incubatedwith the indicated primary antibodies over a range of concentrationsprior to staining with 10 μg/ml APC anti-CD47 and detection by flowcytometry. MFI of the APC signal for each condition is reported.

FIG. 7: SIRPα-rituximab bispecific reagents bind to CD47 with reducedaffinity relative to anti-CD47 and other bispecific antibody formats.Binding of the indicated antibodies to the recombinant CD47 antigen wasmeasured by ELISA over a range of concentrations. Immobilized human CD47tagged with mouse Fc was used to capture the indicated antibodies priorto detection with HRP-conjugated antibody directed against the humankappa light chain. Data are representative of three experimentsperformed in triplicate.

FIG. 8: SIRPα-rituximab bispecific reagents have reduced affinity toCD47 relative to anti-CD47. Kinetic association and dissociationparameters, along with calculated affinity (Kd) were measured by surfaceplasmon resonance using Biacore. The surface was coated with theindicated antibodies via amine-coupling prior to exposure to CD47-Hisanalyte.

FIG. 9: SIRPα-rituximab bispecific reagents bind to CD20 and CD47simultaneously. Schematic of assay to determine simultaneous binding toCD20 and CD47. Double positive events indicate simultaneous binding toCD20 and CD47 by the indicated antibodies.

FIG. 10A-10B: SIRPα-rituximab bispecific reagents bind to CD20 and CD47on dual antigen Raji cells. CD20+CD47+ Raji cells were incubated withthe indicated primary antibodies at 10 μg/ml prior to staining with APCanti-CD47 and detection by flow cytometry. CD20+CD47+ Raji cells wereincubated with the indicated primary antibodies at 10 μg/ml prior tostaining with DyLight 488anti-CD20 and detection by flow cytometry.

FIG. 11A-11D: SIRPα-rituximab bispecific reagents preferentially bind todual antigen tumor cells in the presence of excess CD47-only expressingred blood cells. (FIG. 11A) Schematic of the experimental design toassay for selectivity in binding to dual antigen-expressing cells in thepresence of an excess of CD47-only expressing cells. GFP labeledCD20+CD47+ cells were mixed with a 10-fold excess of CD20−CD47+ humanred blood cells (RBCs). Cell mixtures were incubated with primaryantibody prior to staining with PE anti-human Fcγ secondary and 10 μg/mlAPC anti-CD47, and analyzed by flow cytometry. (FIG. 11B, FIG. 11C)Tumor cells were distinguished from RBCs on the basis of GFP expression.The indicated primary antibodies were used at 10 ug/ml and binding tocells was detected with secondary antibody staining (PE anti-human Fc).2B8=anti-CD20, B6H12=anti-CD47. (FIG. 11D) Binding of APC anti-CD47 tocells is reported as MFI normalized to isotype control.

FIG. 12: SIRPα-rituximab bispecific reagents induce phagocytosis of dualantigen cells. Phagocytosis of CD20+CD47+ Raji-GFP cells by humanmacrophages was assessed by flow cytometry. The percentage of GFP+macrophages was normalized to the maximal response for each donor. Dataaveraged from 3 independent donors and are ±SD.

FIG. 13A-13B: CD20-2GL-SIRPα eliminates lymphoma in vivo. (FIG. 13A) NSGmice transplanted subcutaneously with luciferase-expressing Raji cellswere treated with daily injections of 200 μg mouse IgG control,anti-CD47, SIRPα-Fc, rituximab, CD20-2GL-SIRPα, or 200 μg anti-CD47+200μg rituximab. (n=5 per treatment group). Luciferase imaging was averagedfor all mice in each treatment group. Arrows indicate start (day 7) andstop (day 20) of treatment. Rituximab treatment was compared toCD20-2GL-SIRPα at day 42 (*p<0.05 by t test). (FIG. 13B) Kaplan-Meiersurvival analysis with p-values calculated comparing rituximab singleantibody treatment to combination treatment/CD20-2GL-SIRPα. (*p<0.05 bylog-rank Mantel-Cox test).

FIG. 14A-14D. CD20-2GL-SIRPα HC Reduces Lymphoma Burden and ExtendsSurvival In Vivo. (FIG. 14A) NSG mice were transplanted subcutaneouslywith Raji-luciferase cells. Seven days later, mice were treated with 14daily doses of 200 μg IgG (n=15), SIRPα-Fc (n=15), rituximab (n=15),CD20-2GL-SIRPα HC (n=15) or 200 μg SIRPα-Fc+200 μg rituximab (n=10).Expansion of Raji-luciferase cells was evaluated by bioluminescenceimaging. Each point represents a measurement from an independent mouseand lines indicate median values for each treatment group. p values werederived by t test and compare rituximab to CD20-2GL-SIRPα HC for eachtime point. (FIG. 14B) Kaplan-Meier survival analysis was performed.Arrows indicated start (day 7) and stop (day 21) of treatment.Statistical analysis was performed by Mantel-Cox and compares rituximabto CD20-2GL-SIRPα HC. (FIG. 14C) NSG mice were transplantedintravenously with Raji-luciferase cells. Four days later, mice wereadministered 21 daily doses of antibody treatment as described in (FIG.14A). Each point represents a measurement from an independent mouse(n=10). Lines indicate mean values for each treatment group. p valueswere derived by t test and compare rituximab to CD20-2GL-SIRPα HC foreach time point. (FIG. 14D) Kaplan-Meier survival analysis wasperformed. Arrows indicated start (day 4) and stop (day 25) oftreatment. Statistical analysis was performed by Mantel-Cox and comparesrituximab to CD20-2GL-SIRPα HC.

DETAILED DESCRIPTION OF THE INVENTION

SIRPabodies comprise an immunoglobulin variable region, which mayspecifically bind a tumor antigen, viral antigen, etc., fused to asequence comprising a binding domain of SIRPα. The binding domain ofSIRPα comprises at least the N-terminal Ig-like domain of SIRPα, and mayfurther comprise additional SIRPα sequences. The SIRPabodies find use intherapeutic methods that benefit from the combined activity of blockingCD47 activity, and antibody targeting, e.g. in the treatment of cancer,etc. In some specific embodiments, the SIRPabody comprises anti-CD20activity and a SIRPα binding domain.

Definitions

In the description that follows, a number of terms conventionally usedin the field of cell culture are utilized. In order to provide a clearand consistent understanding of the specification and claims, and thescope to be given to such terms, the following definitions are provided.

The terms “CD47 binding domain agents”, with respect to the interactionbetween SIRPα and its ligand CD47 refer to molecules that prevent thebinding of SIRPα and CD47. For development purposes the binding may beperformed under experimental conditions, e.g. using isolated proteins asbinding partners, using portions of proteins as binding partners, usingyeast display of proteins or portions of proteins as binding partners,and the like.

For therapeutic purposes the binding of SIRPα and CD47 is typically anevent between two cells, where each cell expresses one of the bindingpartners. Of particular interest is the expression of SIRPα onphagocytic cells, such as macrophages; and the expression of CD47 oncells that may be targets for phagocytosis, e.g. tumor cells,circulating hematopoietic cells, and the like. Inhibitors may beidentified using in vitro and in vivo assays for receptor or ligandbinding or signaling.

For the purposes of the invention a blocking agent comprises a SIRPαbinding domain, for example the N-terminal immunoglobulin fold domain ofSEQ ID NO:1, and variants, including without limitation allelicvariants. Relative to the human native SIRPα sequences, (for example seeGenbank accession no. AAH75849, and variants NM_080792.2;XM_005260670.1; XM_005260669.1; NM_001040023.1; NM_001040022.1, hereinspecifically incorporated by reference) a soluble SIRPα binding domainmay comprise the d1 domain of SIRPα, corresponding to residues 31 to 149of the native full-length human protein. In such embodiments, thesoluble SIRPα binding domain may consist of all or a portion of the d1domain; may further comprise one or more amino acids from SIRPα outsideof the d1 domain.

SIRPα binding domains may be at least about 100 amino acids in length,at least about 110, at least about 120, at least about 150, at leastabout 200 amino acids in length, up to the full-length of the wild-typeprotein at the transmembrane domain, i.e. about 343 amino acids inlength, and is optionally fused to a heterologous polypeptide.

A low affinity SIRPα sequence is generally preferred for the purposes ofthe invention, i.e. an affinity equivalent to the affinity of the nativeprotein for binding with CD47. However, in some embodiments an increasedaffinity may be preferred, for which purpose the variants set forth in,for example, WO 2013/109752 (herein specifically incorporated byreference) may be used.

The SIRPα sequence may be a variant of the native human sequence. Asused herein, the term “variant” refers to an entity that showssignificant structural identity with a reference entity but differsstructurally from the reference entity in the presence or level of oneor more chemical moieties as compared with the reference entity. In manyembodiments, a variant also differs functionally from its referenceentity. In general, whether a particular entity is properly consideredto be a “variant” of a reference entity is based on its degree ofstructural identity with the reference entity. As will be appreciated bythose skilled in the art, any biological or chemical reference entityhas certain characteristic structural elements. A variant, bydefinition, is a distinct chemical entity that shares one or more suchcharacteristic structural elements. A polypeptide may have acharacteristic sequence element comprised of a plurality of amino acidshaving designated positions relative to one another in linear orthree-dimensional space and/or contributing to a particular biologicalfunction. For example, a variant polypeptide may differ from a referencepolypeptide as a result of one or more differences in amino acidsequence and/or one or more differences in chemical moieties (e.g.,carbohydrates, lipids, etc) covalently attached to the polypeptidebackbone. In some embodiments, a variant polypeptide shows an overallsequence identity with a reference polypeptide that is at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.Alternatively or additionally, in some embodiments, a variantpolypeptide does not share at least one characteristic sequence elementwith a reference polypeptide. In some embodiments, the referencepolypeptide has one or more biological activities. In some embodiments,a variant polypeptide shares one or more of the biological activities ofthe reference polypeptide. In some embodiments, a variant polypeptidelacks one or more of the biological activities of the referencepolypeptide. In some embodiments, a variant polypeptide shows a reducedlevel of one or more biological activities as compared with thereference polypeptide. In many embodiments, a polypeptide of interest isconsidered to be a “variant” of a parent or reference polypeptide if thepolypeptide of interest has an amino acid sequence that is identical tothat of the parent but for a small number of sequence alterations atparticular positions. Typically, fewer than 20%, 15%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2% of the residues in the variant are substituted ascompared with the parent. In some embodiments, a variant has 10, 9, 8,7, 6, 5, 4, 3, 2, or 1 substituted residue as compared with a parent.Often, a variant has a very small number (e.g., fewer than 5, 4, 3, 2,or 1) number of substituted functional residues (i.e., residues thatparticipate in a particular biological activity). Furthermore, a varianttypically has not more than 5, 4, 3, 2, or 1 additions or deletions, andoften has no additions or deletions, as compared with the parent.Moreover, any additions or deletions are typically fewer than about 25,about 20, about 19, about 18, about 17, about 16, about 15, about 14,about 13, about 10, about 9, about 8, about 7, about 6, and commonly arefewer than about 5, about 4, about 3, or about 2 residues. In someembodiments, the parent or reference polypeptide is one found in nature.As will be understood by those of ordinary skill in the art, a pluralityof variants of a particular polypeptide of interest may commonly befound in nature, particularly when the polypeptide of interest is aninfectious agent polypeptide.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms also apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an .alpha. carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a mammal being assessed for treatmentand/or being treated. In an embodiment, the mammal is a human. The terms“subject,” “individual,” and “patient” encompass, without limitation,individuals having cancer. Subjects may be human, but also include othermammals, particularly those mammals useful as laboratory models forhuman disease, e.g. mouse, rat, etc. Also included are mammals such asdomestic and other species of canines, felines, and the like.

The terms “cancer,” “neoplasm,” and “tumor” are used interchangeablyherein to refer to cells which exhibit autonomous, unregulated growth,such that they exhibit an aberrant growth phenotype characterized by asignificant loss of control over cell proliferation. Cells of interestfor detection, analysis, or treatment in the present application includeprecancerous (e.g., benign), malignant, pre-metastatic, metastatic, andnon-metastatic cells. Cancers of virtually every tissue are known. Thephrase “cancer burden” refers to the quantum of cancer cells or cancervolume in a subject. Reducing cancer burden accordingly refers toreducing the number of cancer cells or the cancer volume in a subject.The term “cancer cell” as used herein refers to any cell that is acancer cell or is derived from a cancer cell e.g. clone of a cancercell. Many types of cancers are known to those of skill in the art,including solid tumors such as carcinomas, sarcomas, glioblastomas,melanomas, lymphomas, myelomas, etc., and circulating cancers such asleukemias. Examples of cancer include but are not limited to, ovariancancer, breast cancer, colon cancer, lung cancer, prostate cancer,hepatocellular cancer, gastric cancer, pancreatic cancer, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, cancer of theurinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, headand neck cancer, and brain cancer.

The “pathology” of cancer includes all phenomena that compromise thewell-being of the patient. This includes, without limitation, abnormalor uncontrollable cell growth, metastasis, interference with the normalfunctioning of neighboring cells, release of cytokines or othersecretory products at abnormal levels, suppression or aggravation ofinflammatory or immunological response, neoplasia, premalignancy,malignancy, invasion of surrounding or distant tissues or organs, suchas lymph nodes, etc.

As used herein, the terms “cancer recurrence” and “tumor recurrence,”and grammatical variants thereof, refer to further growth of neoplasticor cancerous cells after diagnosis of cancer. Particularly, recurrencemay occur when further cancerous cell growth occurs in the canceroustissue. “Tumor spread,” similarly, occurs when the cells of a tumordisseminate into local or distant tissues and organs; therefore tumorspread encompasses tumor metastasis. “Tumor invasion” occurs when thetumor growth spread out locally to compromise the function of involvedtissues by compression, destruction, or prevention of normal organfunction.

As used herein, the term “metastasis” refers to the growth of acancerous tumor in an organ or body part, which is not directlyconnected to the organ of the original cancerous tumor. Metastasis willbe understood to include micrometastasis, which is the presence of anundetectable amount of cancerous cells in an organ or body part which isnot directly connected to the organ of the original cancerous tumor.Metastasis can also be defined as several steps of a process, such asthe departure of cancer cells from an original tumor site, and migrationand/or invasion of cancer cells to other parts of the body.

Non-Hodgkin lymphomas (NHL) are a heterogeneous group of disordersinvolving malignant monoclonal proliferation of lymphoid cells inlymphoreticular sites, including lymph nodes, bone marrow, the spleen,the liver, and the gastrointestinal tract. Presenting symptoms usuallyinclude peripheral lymphadenopathy. Compared with Hodgkin lymphoma,there is a greater likelihood of disseminated disease at the time ofdiagnosis. However, NHL is not one disease but rather a category oflymphocyte malignancies. These types can be divided into aggressive(fast-growing) and indolent (slow-growing) types, and they can be formedfrom either B-cells or T-cells. B-cell non-Hodgkin lymphomas includeBurkitt lymphoma, chronic lymphocytic leukemia/small lymphocyticlymphoma (CLL/SLL), diffuse large B-cell lymphoma, follicular lymphoma,precursor B-lymphoblastic lymphoma, and mantle cell lymphoma, amongothers. T-cell non-Hodgkin lymphomas include mycosis fungoides,anaplastic large cell lymphoma, and precursor T-lymphoblastic lymphoma.Lymphomas that occur after bone marrow or stem cell transplantation areusually B-cell non-Hodgkin lymphomas. Prognosis and treatment depend onthe stage and type of disease.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype ofnon-Hodgkin lymphoma (NHL), accounting for approximately 30% of allnewly diagnosed cases and more than 80% of aggressive lymphomas. Recentinsights into the pathogenesis of DLBCL suggest that it is aheterogeneous group of B-cell lymphomas rather than a singleclinicopathologic entity. Multiple histologic subtypes and morphologicvariants are recognized, a variety of molecular and geneticabnormalities are variably present, and patients exhibit a wide range ofclinical presentations and outcomes. Gene-expression profiling studieshave identified at least 3 distinct molecular subtypes of DLBCL, onewith an expression profile similar to normal germinal center B cells(GCB subtype), one mimicking activated peripheral-blood B cells (ABCsubtype), and a third, primary mediastinal large B-cell lymphoma(PMBCL), typically presenting with mediastinal lymphadenopathy anddisplaying some molecular genetic similarities to Hodgkin lymphoma. Asmall number of cases do not fit into any of these categories and havebeen designated as “unclassifiable.”

The term “sample” with respect to a patient encompasses blood and otherliquid samples of biological origin, solid tissue samples such as abiopsy specimen or tissue cultures or cells derived therefrom and theprogeny thereof. The definition also includes samples that have beenmanipulated in any way after their procurement, such as by treatmentwith reagents; washed; or enrichment for certain cell populations, suchas cancer cells. The definition also includes sample that have beenenriched for particular types of molecules, e.g., nucleic acids,polypeptides, etc. The term “biological sample” encompasses a clinicalsample, and also includes tissue obtained by surgical resection, tissueobtained by biopsy, cells in culture, cell supernatants, cell lysates,tissue samples, organs, bone marrow, blood, plasma, serum, and the like.A “biological sample” includes a sample obtained from a patient's cancercell, e.g., a sample comprising polynucleotides and/or polypeptides thatis obtained from a patient's cancer cell (e.g., a cell lysate or othercell extract comprising polynucleotides and/or polypeptides); and asample comprising cancer cells from a patient. A biological samplecomprising a cancer cell from a patient can also include non-cancerouscells.

The term “diagnosis” is used herein to refer to the identification of amolecular or pathological state, disease or condition, such as theidentification of a molecular subtype of breast cancer, prostate cancer,or other type of cancer.

The term “prognosis” is used herein to refer to the prediction of thelikelihood of cancer-attributable death or progression, includingrecurrence, metastatic spread, and drug resistance, of a neoplasticdisease, such as ovarian cancer. The term “prediction” is used herein torefer to the act of foretelling or estimating, based on observation,experience, or scientific reasoning. In one example, a physician maypredict the likelihood that a patient will survive, following surgicalremoval of a primary tumor and/or chemotherapy for a certain period oftime without cancer recurrence.

As used herein, the terms “treatment,” “treating,” and the like, referto administering an agent, or carrying out a procedure, for the purposesof obtaining an effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of effecting a partial or complete cure fora disease and/or symptoms of the disease. “Treatment,” as used herein,may include treatment of a tumor in a mammal, particularly in a human,and includes: (a) preventing the disease or a symptom of a disease fromoccurring in a subject which may be predisposed to the disease but hasnot yet been diagnosed as having it (e.g., including diseases that maybe associated with or caused by a primary disease; (b) inhibiting thedisease, i.e., arresting its development; and (c) relieving the disease,i.e., causing regression of the disease.

Treating may refer to any indicia of success in the treatment oramelioration or prevention of an cancer, including any objective orsubjective parameter such as abatement; remission; diminishing ofsymptoms or making the disease condition more tolerable to the patient;slowing in the rate of degeneration or decline; or making the finalpoint of degeneration less debilitating. The treatment or ameliorationof symptoms can be based on objective or subjective parameters;including the results of an examination by a physician. Accordingly, theterm “treating” includes the administration of the compounds or agentsof the present invention to prevent or delay, to alleviate, or to arrestor inhibit development of the symptoms or conditions associated withocular disease. The term “therapeutic effect” refers to the reduction,elimination, or prevention of the disease, symptoms of the disease, orside effects of the disease in the subject.

“In combination with”, “combination therapy” and “combination products”refer, in certain embodiments, to the concurrent administration to apatient of a first therapeutic and the compounds as used herein. Whenadministered in combination, each component can be administered at thesame time or sequentially in any order at different points in time.Thus, each component can be administered separately but sufficientlyclosely in time so as to provide the desired therapeutic effect.

In some embodiments, treatment is accomplished by administering aSIRPabody in combination with a cytotoxic agent. One exemplary class ofcytotoxic agents are chemotherapeutic agents. Exemplary chemotherapeuticagents include, but are not limited to, aldesleukin, altretamine,amifostine, asparaginase, bleomycin, capecitabine, carboplatin,carmustine, cladribine, cisapride, cisplatin, cyclophosphamide,cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin,dronabinol, duocarmycin, epoetin alpha, etoposide, filgrastim,fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea,idarubicin, ifosfamide, interferon alpha, irinotecan, lansoprazole,levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide,mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel(Taxol™), pilocarpine, prochloroperazine, rituximab, saproin, tamoxifen,taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristineand vinorelbine tartrate.

“Concomitant administration” of a known cancer therapeutic drug with apharmaceutical composition of the present invention means administrationof the drug and SIRPabody at such time that both the known drug and thecomposition of the present invention will have a therapeutic effect.Such concomitant administration may involve concurrent (i.e. at the sametime), prior, or subsequent administration of the drug with respect tothe administration of a compound of the invention. A person of ordinaryskill in the art would have no difficulty determining the appropriatetiming, sequence and dosages of administration for particular drugs andcompositions of the present invention.

As used herein, the phrase “disease-free survival,” refers to the lackof such tumor recurrence and/or spread and the fate of a patient afterdiagnosis, with respect to the effects of the cancer on the life-span ofthe patient. The phrase “overall survival” refers to the fate of thepatient after diagnosis, despite the possibility that the cause of deathin a patient is not directly due to the effects of the cancer. Thephrases, “likelihood of disease-free survival”, “risk of recurrence” andvariants thereof, refer to the probability of tumor recurrence or spreadin a patient subsequent to diagnosis of cancer, wherein the probabilityis determined according to the process of the invention.

As used herein, the term “correlates,” or “correlates with,” and liketerms, refers to a statistical association between instances of twoevents, where events include numbers, data sets, and the like. Forexample, when the events involve numbers, a positive correlation (alsoreferred to herein as a “direct correlation”) means that as oneincreases, the other increases as well. A negative correlation (alsoreferred to herein as an “inverse correlation”) means that as oneincreases, the other decreases.

“Dosage unit” refers to physically discrete units suited as unitarydosages for the particular individual to be treated. Each unit cancontain a predetermined quantity of active compound(s) calculated toproduce the desired therapeutic effect(s) in association with therequired pharmaceutical carrier. The specification for the dosage unitforms can be dictated by (a) the unique characteristics of the activecompound(s) and the particular therapeutic effect(s) to be achieved, and(b) the limitations inherent in the art of compounding such activecompound(s).

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

“Pharmaceutically acceptable salts and esters” means salts and estersthat are pharmaceutically acceptable and have the desiredpharmacological properties. Such salts include salts that can be formedwhere acidic protons present in the compounds are capable of reactingwith inorganic or organic bases. Suitable inorganic salts include thoseformed with the alkali metals, e.g. sodium and potassium, magnesium,calcium, and aluminum. Suitable organic salts include those formed withorganic bases such as the amine bases, e.g., ethanolamine,diethanolamine, triethanolamine, tromethamine, N methylglucamine, andthe like. Such salts also include acid addition salts formed withinorganic acids (e.g., hydrochloric and hydrobromic acids) and organicacids (e.g., acetic acid, citric acid, maleic acid, and the alkane- andarene-sulfonic acids such as methanesulfonic acid and benzenesulfonicacid). Pharmaceutically acceptable esters include esters formed fromcarboxy, sulfonyloxy, and phosphonoxy groups present in the compounds,e.g., C₁₋₆ alkyl esters. When there are two acidic groups present, apharmaceutically acceptable salt or ester can be a mono-acid-mono-saltor ester or a di-salt or ester; and similarly where there are more thantwo acidic groups present, some or all of such groups can be salified oresterified. Compounds named in this invention can be present inunsalified or unesterified form, or in salified and/or esterified form,and the naming of such compounds is intended to include both theoriginal (unsalified and unesterified) compound and its pharmaceuticallyacceptable salts and esters. Also, certain compounds named in thisinvention may be present in more than one stereoisomeric form, and thenaming of such compounds is intended to include all single stereoisomersand all mixtures (whether racemic or otherwise) of such stereoisomers.

The terms “pharmaceutically acceptable”, “physiologically tolerable” andgrammatical variations thereof, as they refer to compositions, carriers,diluents and reagents, are used interchangeably and represent that thematerials are capable of administration to or upon a human without theproduction of undesirable physiological effects to a degree that wouldprohibit administration of the composition.

As used herein, the term “antibody” refers to a polypeptide thatincludes canonical immunoglobulin sequence elements sufficient to conferspecific binding to a particular target antigen. As is known in the art,intact antibodies as produced in nature are approximately 150 kDtetrameric agents comprised of two identical heavy chain polypeptides(about 50 kD each) and two identical light chain polypeptides (about 25kD each) that associate with each other into what is commonly referredto as a “Y-shaped” structure. Each heavy chain is comprised of at leastfour domains (each about 110 amino acids long)—an amino-terminalvariable (VH) domain (located at the tips of the Y structure), followedby three constant domains: CH1, CH2, and the carboxy-terminal CH3(located at the base of the Y's stem). A short region, known as the“switch”, connects the heavy chain variable and constant regions. The“hinge” connects CH2 and CH3 domains to the rest of the antibody. Twodisulfide bonds in this hinge region connect the two heavy chainpolypeptides to one another in an intact antibody. Each light chain iscomprised of two domains—an amino-terminal variable (VL) domain,followed by a carboxy-terminal constant (CL) domain, separated from oneanother by another “switch”. Intact antibody tetramers are comprised oftwo heavy chain-light chain dimers in which the heavy and light chainsare linked to one another by a single disulfide bond; two otherdisulfide bonds connect the heavy chain hinge regions to one another, sothat the dimers are connected to one another and the tetramer is formed.Naturally-produced antibodies are also glycosylated, typically on theCH2 domain.

Each domain in a natural antibody has a structure characterized by an“immunoglobulin fold” formed from two beta sheets (e.g., 3-, 4-, or5-stranded sheets) packed against each other in a compressedantiparallel beta barrel. Each variable domain contains threehypervariable loops known as “complement determining regions” (CDR1,CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1,FR2, FR3, and FR4). When natural antibodies fold, the FR regions formthe beta sheets that provide the structural framework for the domains,and the CDR loop regions from both the heavy and light chains arebrought together in three-dimensional space so that they create a singlehypervariable antigen binding site located at the tip of the Ystructure. The Fc region of naturally-occurring antibodies binds toelements of the complement system, and also to receptors on effectorcells, including for example effector cells that mediate cytotoxicity.

As is known in the art, affinity and/or other binding attributes of Fcregions for Fc receptors can be modulated through glycosylation or othermodification. In some embodiments, SIRPabodies produced and/or utilizedin accordance with the present invention may include glycosylated Fcdomains, including Fc domains with modified or engineered suchglycosylation. For purposes of the present invention, in certainembodiments, any polypeptide or complex of polypeptides that includessufficient immunoglobulin domain sequences as found in naturalantibodies can be referred to and/or used as an “antibody”, whether suchpolypeptide is naturally produced (e.g., generated by an organismreacting to an antigen), or produced by recombinant engineering,chemical synthesis, or other artificial system or methodology.

In some embodiments, an antibody has constant region sequences that arecharacteristic of mouse, rabbit, primate, or human antibodies. In someembodiments, antibody sequence elements are humanized, primatized,chimeric, etc, as is known in the art. Moreover, the term “antibody” asused herein, can refer in appropriate embodiments (unless otherwisestated or clear from context) to any of the art-known or developedconstructs or formats for utilizing antibody structural and functionalfeatures in alternative presentation. For example, embodiments, anantibody utilized in accordance with the present invention is in aformat selected from, but not limited to, intact IgG, IgE and IgM, bi-or multi-specific antibodies (e.g., Zybodies®, etc), single chain Fvs,polypeptide-Fc fusions, Fabs, cameloid antibodies, masked antibodies(e.g., Probodies®), Small Modular ImmunoPharmaceuticals (“SMIPs™”),single chain or Tandem diabodies (TendAb®), VHHs, Anticalins®,Nanobodies®, minibodies, BiTE®s, ankyrin repeat proteins or DARPINs®,Avimers®, a DART, a TCR-like antibody, Adnectins®, Affilins®,Trans-bodies®, Affibodies®, a TrimerX®, MicroProteins, Fynomers®,Centyrins®, and a KALBITOR®.

As used herein, the term “antibody-dependent cellular cytotoxicity” or“ADCC” refers to a phenomenon in which target cells bound by antibodyare killed by immune effector cells. Without wishing to be bound by anyparticular theory, ADCC is typically understood to involve Fc receptor(FcR)-bearing effector cells can recognizing and subsequently killingantibody-coated target cells (e.g., cells that express on their surfacespecific antigens to which an antibody is bound). Effector cells thatmediate ADCC can include immune cells, including but not limited to oneor more of natural killer (NK) cells, macrophage, neutrophils,eosinophils.

The term “antigen”, as used herein, refers to an agent that elicits animmune response; and/or for the purposes of the invention, (ii) an agentthat binds to an antibody. In general, an antigen may be or include anychemical entity such as, for example, a small molecule, a nucleic acid,a polypeptide, a carbohydrate, a lipid, a polymer (in some embodimentsother than a biologic polymer [e.g., other than a nucleic acid or aminoacid polymer) etc. In some embodiments, an antigen is or comprises apolypeptide. In some embodiments, an antigen is or comprises a glycan.Those of ordinary skill in the art will appreciate that, in general, anantigen may be provided in isolated or pure form, or alternatively maybe provided in crude form (e.g., together with other materials, forexample in an extract such as a cellular extract or other relativelycrude preparation of an antigen-containing source).

The term “anti-CD20 antibody” refers herein to monoclonal or polyclonalantibodies with specificity for the polypeptide CD20. Antibodies withspecificity for CD20 can be prepared by methods that are well understoodin the art. Preferred antibody compositions are ones that have beenselected for antibodies directed against a polypeptide or polypeptidesof CD20. Particularly preferred polyclonal antibody preparations areones that contain only antibodies directed against a polypeptide orpolypeptides of CD20. Anti-CD20 antibodies that are suitable for use inthe current invention would include, for example, rituximab, ibritumomabtiuxetan, tositumomab, AME-133v (Applied Molecular Evolution),Ocrelizumab (Roche), Ofatumumab (Genmab), TRU-015 (Trubion) and IMMU-106(Immunomedics). In one embodiment, the anti-CD20 antibody is rituximab.

“CD20”, “CD20 protein”, and “CD20 polypeptide” are used interchangeablyherein to refer to a polypeptide encoded by a member of themembrane-spanning 4A gene family. This gene, referred to as “MS4A1”,“Membrane-spanning 4-domains, subfamily A, member 1”, “B1” and“B-lymphocyte surface antigen B1”, encodes a non-glycosylatedphosphoprotein of 297 amino acids, as described at, for example, GenbankNM_152866 and Genbank NM_021950 (alternative splice variants that encodethe same protein). CD20 polypeptide is expressed on the surface of Bcells beginning at the late pre-B cell phase of development, and plays arole in B cell proliferation.

Antibodies targeting tumor antigens have been approved for use intreating cancers, and are rapidly becoming standard of care. Anon-comprehensive list of certain human antigens targeted by known,available antibody agents, and notes certain cancer indications forwhich the antibody agents have been proposed to be useful:

CD2 Siplizumab Non-Hodgkin's Lymphoma CD3 UCHT1 Peripheral or CutaneousT-cell CD4 HuMax-CD4 Lymphoma CD19 SAR3419, MEDI-551 Diffuse LargeB-cell Lymphoma CD19 and CD3 or Bispecific antibodies such asNon-Hodgkin's Lymphoma CD22 Blinatumomab, DT2219ARL CD20 Rituximab,Veltuzumab, B cell malignancies (Non-Hodgkin's Tositumomab, Ofatumumab,lymphoma, Chronic lymphocytic Ibritumomab, Obinutuzumab, leukemia) CD22(SIGLEC2) Inotuzumab, tetraxetan, CAT- Chemotherapy-resistant hairy cell8015, DCDT2980S, leukemia, Hodgkin's lymphoma Bectumomab CD30Brentuximab vedotin CD33 Gemtuzumab ozogamicin Acute myeloid leukemia(Mylotarg) CD37 TRU-016 Chronic lymphocytic leukemia CD38 DaratumumabMultiple myeloma, hematological tumors CD40 Lucatumumab Non-Hodgkin'slymphoma CD52 Alemtuzumab (Campath) Chronic lymphocytic leukemia CD56(NCAM1) Lorvotuzumab Small Cell Lung Cancer CD66e (CEA) LabetuzumabBreast, colon and lung tumors CD70 SGN-75 Non-Hodgkin's lymphoma CD74Milatuzumab Non-Hodgkin's lymphoma CD138 (SYND1) BT062 Multiple MyelomaCD152 (CTLA-4) Ipilimumab Metastatic melanoma CD221 (IGF1R) AVE1642,IMC-A12, MK-0646, Glioma, lung, breast, head and neck, R150, CP 751871prostate and thyroid cancer CD254 (RANKL) Denosumab Breast and prostatecarcinoma CD261 (TRAILR1) Mapatumumab Colon, lung and pancreas tumorsand CD262 (TRAILR2) HGS-ETR2, CS-1008 haematological malignancies CD326(Epcam) Edrecolomab, 17-1A, IGN101, Colon and rectal cancer, malignantCatumaxomab, ascites, epithelial tumors (breast, Adecatumumab colon,lung) CD309 (VEGFR2) IM-2C6, CDP791 Epithelium-derived solid tumorsCD319 (SLAMF7) HuLuc63 Multiple myeloma CD340 (HER2) Trastuzumab,Pertuzumab, Breast cancer Ado-trastuzumab emtansine CAIX (CA9) cG250Renal cell carcinoma EGFR (c-erbB) Cetuximab, Panitumumab, Solid tumorsincluding glioma, lung, nimotuzumab and 806 breast, colon, and head andneck tumors EPHA3 (HEK) KB004, IIIA4 Lung, kidney and colon tumors,melanoma, glioma and haematological malignancies Episialin EpitumomabEpithelial ovarian tumors FAP Sibrotuzumab and F19 Colon, breast, lung,pancreas, and head and neck tumors HLA-DR beta Apolizumab Chroniclymphocytic leukemia, non- Hodkin's lymphoma FOLR-1 Farletuzumab Ovariantumors 5T4 Anatumomab Non-small cell lung cancer GD3/GD2 3F8, ch14.18,KW-2871 Neuroectodermal and epithelial tumors gpA33 huA33 Colorectalcarcinoma GPNMB Glembatumumab Breast cancer HER3 (ERBB3) MM-121 Breast,colon, lung, ovarian, and prostate tumors Integrin αVβ3 EtaracizumabTumor vasculature Integrin α5β1 Volociximab Tumor vasculature Lewis-Yantigen hu3S193, IgN311 Breast, colon, lung and prostate tumors MET(HGFR) AMG 102, METMAB, Breast, ovary and lung tumors SCH900105Mucin-1/CanAg Pemtumomab, oregovomab, Breast, colon, lung and ovariantumors Cantuzumab PSMA ADC, J591 Prostate Cancer PhosphatidylserineBavituximab Solid tumors TAG-72 Minretumomab Breast, colon and lungtumors Tenascin 81C6 Glioma, breast and prostate tumours VEGFBevacizumab Tumour vasculature

As used herein, the term “therapeutically effective amount” means anamount that is sufficient, when administered to a population sufferingfrom or susceptible to a disease, disorder, and/or condition inaccordance with a therapeutic dosing regimen, to treat the disease,disorder, and/or condition. In some embodiments, a therapeuticallyeffective amount is one that reduces the incidence and/or severity of,stabilizes one or more characteristics of, and/or delays onset of, oneor more symptoms of the disease, disorder, and/or condition. Those ofordinary skill in the art will appreciate that the term “therapeuticallyeffective amount” does not in fact require successful treatment beachieved in a particular individual. Rather, a therapeutically effectiveamount may be that amount that provides a particular desiredpharmacological response in a significant number of subjects whenadministered to patients in need of such treatment.

For example, in some embodiments, the term “therapeutically effectiveamount”, refers to an amount which, when administered to an individualin need thereof in the context of inventive therapy, will block,stabilize, attenuate, or reverse a cancer-supportive process occurringin said individual, or will enhance or increase a cancer-suppressiveprocess in said individual. In the context of cancer treatment, a“therapeutically effective amount” is an amount which, when administeredto an individual diagnosed with a cancer, will prevent, stabilize,inhibit, or reduce the further development of cancer in the individual.A particularly preferred “therapeutically effective amount” of acomposition described herein reverses (in a therapeutic treatment) thedevelopment of a malignancy, such as a B cell lymphoma, or helps achieveor prolong remission of a malignancy.

A therapeutically effective amount administered to an individual totreat a cancer in that individual may be the same or different from atherapeutically effective amount administered to promote remission orinhibit metastasis. As with most cancer therapies, the therapeuticmethods described herein are not to be interpreted as, restricted to, orotherwise limited to a “cure” for cancer; rather the methods oftreatment are directed to the use of the described compositions to“treat” a cancer, i.e., to effect a desirable or beneficial change inthe health of an individual who has cancer. Such benefits are recognizedby skilled healthcare providers in the field of oncology and include,but are not limited to, a stabilization of patient condition, a decreasein tumor size (tumor regression), an improvement in vital functions(e.g., improved function of cancerous tissues or organs), a decrease orinhibition of further metastasis, a decrease in opportunisticinfections, an increased survivability, a decrease in pain, improvedmotor function, improved cognitive function, improved feeling of energy(vitality, decreased malaise), improved feeling of well-being,restoration of normal appetite, restoration of healthy weight gain, andcombinations thereof.

In addition, regression of a particular tumor in an individual (e.g., asthe result of treatments described herein) may also be assessed bytaking samples of cancer cells from the site of a tumor such as a B celllymphoma (e.g., over the course of treatment) and testing the cancercells for the level of metabolic and signaling markers to monitor thestatus of the cancer cells to verify at the molecular level theregression of the cancer cells to a less malignant phenotype. Forexample, tumor regression induced by employing the methods of thisinvention would be indicated by finding a decrease in any of thepro-angiogenic markers, an increase in anti-angiogenic markers, thenormalization (i.e., alteration toward a state found in normalindividuals not suffering from cancer) of metabolic pathways,intercellular signaling pathways, or intracellular signaling pathwaysthat exhibit abnormal activity in individuals diagnosed with cancer.Those of ordinary skill in the art will appreciate that, in someembodiments, a therapeutically effective amount may be formulated and/oradministered in a single dose. In some embodiments, a therapeuticallyeffective amount may be formulated and/or administered in a plurality ofdoses, for example, as part of a dosing regimen.

Methods of Use

The present invention provides methods for treating, reducing orpreventing cancer, including without limitation hematopoietic cancers,and metastasis of cancers, by inhibiting the interaction between SIRPαand CD47 in a targeted manner, thereby increasing phagocytosis of tumorcells. Such methods include administering to a subject in need oftreatment a therapeutically effective amount or an effective dose of aSIRPabody, where there immunoglobulin variable region of the SIRPabodyspecifically binds a cell surface protein on the tumor cell,particularly binding to CD20. Effective doses of the therapeutic entityof the present invention, e.g. for the treatment of cancer, varydepending upon many different factors, including means ofadministration, target site, physiological state of the patient, whetherthe patient is human or an animal, other medications administered, andwhether treatment is prophylactic or therapeutic. Usually, the patientis a human, but nonhuman mammals may also be treated. Treatment dosagesneed to be titrated to optimize safety and efficacy.

In some embodiments, the dosage may range from about 0.0001 to 100mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. Forexample dosages can be 1 mg/kg body weight or 10 mg/kg body weight orwithin the range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once every two weeks or once a month or once every 3 to 6months. Therapeutic entities of the present invention are usuallyadministered on multiple occasions. Intervals between single dosages canbe weekly, monthly or yearly. Intervals can also be irregular asindicated by measuring blood levels of the therapeutic entity in thepatient. Alternatively, therapeutic entities of the present inventioncan be administered as a sustained release formulation, in which caseless frequent administration is required. Dosage and frequency varydepending on the half-life of the polypeptide in the patient.

In prophylactic applications, a relatively low dosage may beadministered at relatively infrequent intervals over a long period oftime. Some patients continue to receive treatment for the rest of theirlives. In other therapeutic applications, a relatively high dosage atrelatively short intervals is sometimes required until progression ofthe disease is reduced or terminated, and preferably until the patientshows partial or complete amelioration of symptoms of disease.Thereafter, the patent can be administered a prophylactic regime.

In still other embodiments, methods of the present invention includetreating, reducing or preventing tumor growth, tumor metastasis or tumorinvasion of cancers including lymphomas, leukemias, carcinomas,melanomas, glioblastomas, sarcomas, myelomas, etc. For prophylacticapplications, pharmaceutical compositions or medicaments areadministered to a patient susceptible to, or otherwise at risk ofdisease in an amount sufficient to eliminate or reduce the risk, lessenthe severity, or delay the outset of the disease, including biochemical,histologic and/or behavioral symptoms of the disease, its complicationsand intermediate pathological phenotypes presenting during developmentof the disease.

Compositions for the treatment of cancer can be administered byparenteral, topical, intravenous, intratumoral, oral, subcutaneous,intraarterial, intracranial, intraperitoneal, intranasal orintramuscular means. A typical route of administration is intravenous orintratumoral, although other routes can be equally effective.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above. Langer, Science 249: 1527,1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. Theagents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient. The pharmaceutical compositions are generally formulated assterile, substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Toxicity of the proteins described herein can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., by determining the LD₅₀ (the dose lethal to 50% of the population)or the LD₁₀₀ (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index. The dataobtained from these cell culture assays and animal studies can be usedin formulating a dosage range that is not toxic for use in human. Thedosage of the proteins described herein lies preferably within a rangeof circulating concentrations that include the effective dose withlittle or no toxicity. The dosage can vary within this range dependingupon the dosage form employed and the route of administration utilized.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition.

In one aspect, the invention provides SIRPabody polypeptides, andisolated nucleic acids encoding SIRPabody polypeptides. For recombinantproduction of the SIRPabody, the nucleic acid encoding it is insertedinto a replicable vector for further cloning (amplification of the DNA)or for expression. DNA encoding the monoclonal antibody is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the antibody). Many vectors areavailable. The vector components generally include, but are not limitedto, one or more of the following: a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence.

The SIRPabody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for human γ3 (Guss et al., EMBO J. 5:15671575 (1986)). Thematrix to which the affinity ligand is attached is most often agarose,but other matrices are available. Mechanically stable matrices such ascontrolled pore glass or poly(styrenedivinyl)benzene allow for fasterflow rates and shorter processing times than can be achieved withagarose. Where the antibody comprises a CH₃ domain, the Bakerbond ABX™resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.Other techniques for protein purification such as fractionation on anion-exchange column, ethanol precipitation, Reverse Phase HPLC,chromatography on silica, chromatography on heparin SEPHAROSE™chromatography on an anion or cation exchange resin (such as apolyaspartic acid column), chromatofocusing, SDS-PAGE, and ammoniumsulfate precipitation are also available depending on the antibody to berecovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Therapeutic formulations comprising one or more SIRPabodies of theinvention are prepared for storage by mixing the SIRPabody having thedesired degree of purity with optional physiologically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. The antibody composition will beformulated, dosed, and administered in a fashion consistent with goodmedical practice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The “therapeutically effective amount”of the antibody to be administered will be governed by suchconsiderations, and is the minimum amount necessary to prevent the CD47associated disease.

The therapeutic dose may be at least about 0.01 μg/kg body weight, atleast about 0.05 μg/kg body weight; at least about 0.1 μg/kg bodyweight, at least about 0.5 μg/kg body weight, at least about 1 μg/kgbody weight, at least about 2.5 μg/kg body weight, at least about 5μg/kg body weight, and not more than about 100 μg/kg body weight. Itwill be understood by one of skill in the art that such guidelines willbe adjusted for the molecular weight of the active agent, e.g. in theuse of antibody fragments, or in the use of antibody conjugates. Thedosage may also be varied for localized administration, e.g. intranasal,inhalation, etc., or for systemic administration, e.g. i.m., i.p., i.v.,and the like.

The antibody need not be, but is optionally formulated with one or moreagents that potentiate activity, or that otherwise increase thetherapeutic effect. These are generally used in the same dosages andwith administration routes as used hereinbefore or about from 1 to 99%of the heretofore employed dosages.

Acceptable carriers, excipients, or stabilizers are non-toxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Formulations to be used for in vivo administration must be sterile. Thisis readily accomplished by filtration through sterile filtrationmembranes.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label. Suitable containers include, for example, bottles, vials,syringes, and test tubes. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is effective for treating the condition and may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The active agent in the composition is the SIRPabody. The labelon, or associated with, the container indicates that the composition isused for treating the condition of choice. The article of manufacturemay further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

Also within the scope of the invention are kits comprising thecompositions (e.g., SIRPabodies and formulations thereof) of theinvention and instructions for use. The kit can further contain a leastone additional reagent, e.g. a chemotherapeutic drug, etc. Kitstypically include a label indicating the intended use of the contents ofthe kit. The term label includes any writing, or recorded materialsupplied on or with the kit, or which otherwise accompanies the kit.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that various changes and modifications can bemade without departing from the spirit or scope of the invention.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. For example, due to codon redundancy, changescan be made in the underlying DNA sequence without affecting the proteinsequence. Moreover, due to biological functional equivalencyconsiderations, changes can be made in protein structure withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

Example 1

SIRPabodies were created with the aim of co-targeting CD47 and a secondantigen. CD20 was chosen for this purpose. The N-terminal immunoglobulinfold from wild type human SIRPα was engineered onto either the N or Cterminus of the heavy chain of rituximab, an established anti-CD20antibody (FIG. 1A, 1B).

Production of recombinant SIRPα-rituximab bispecific reagents wasconfirmed by analysis of purified protein on reducing and non-reducingSDS-PAGE (FIG. 2).

Each SIRPabody (engineered bispecific variant) retained the ability tobind the CD20 antigen expressed on the cell surface (FIG. 3).Accordingly, when CD20-expressing cells were stained with SIRPabodiesprior to incubation with DyLight 488anti-CD20, all antibodies masked theCD20 epitope preventing subsequent binding with DyLight 488anti-CD20(FIG. 4).

All SIRPabody reagents were able to bind to CD47, although the strengthof binding varied between formats (FIG. 5).

When CD47-expressing cells were first stained with SIRPabodies,subsequent incubation with labeled anti-CD47 outcompeted the primarySIRPabody staining, indicating a weak affinity of each SIRPabodies forCD47 (FIG. 6). Weak binding to CD47 is a desired characteristic forSIRPabodies, as those reagents will require the avidity contributionsfrom interactions with CD20 for binding to dual antigen cells.

To further explore the affinity of SIRPabodies to CD47, ELISA assayswere performed to measure binding to recombinant CD47 antigen (FIG. 7).Consistently, all variants exhibited reduced affinity for CD47 relativeto monoclonal anti-CD47. The affinity of each SIRPabody for CD47 wascalculated using surface plasmon resonance measurements and relativeaffinities were consistent with all previous assays, indicating areduced affinity for CD47 compared to monoclonal anti-CD47 (FIG. 8).

To assess whether SIRPabodies are capable of simultaneous binding toCD20 and CD47, antibodies were coincubated with CD20+CD47− cells andfluorescent NeutrAvidin coated with biotinylated CD47 fusion protein.SIRPabody binding was detected with secondary antibody and doublepositive fluorescent events indicated simultaneous binding to eachantigen by the primary antibody (FIG. 9). Binding to both CD20 and CD47on the cell surface was demonstrated by incubating cells expressing bothantigens with SIRPabodies prior to staining with APC anti-CD47 orDyLight 488anti-CD20. All bispecific reagents blocked the subsequentbinding of labeled antibody indicating binding to both antigens by theprimary antibody stain (FIG. 10).

To determine whether the SIRPabodies have the desired selectivity fordual antigen cells in the presence of challenge with excess CD47-onlyexpressing cells, CD20+CD47+ Raji cells were mixed with 20-fold excessred blood cells (RBCs) (CD47+CD20−). Cell mixtures were stained withSIRPabodies and antibody binding was detected with PE anti-humansecondary (FIG. 11). All SIRPabodies bound to dual antigen tumor cells,while binding to single antigen RBCs was absent. Moreover, binding todual antigen tumor cells was in part mediated by binding to CD47 asindicated by the ability of primary antibody stain to block subsequentstaining with APC anti-CD47 (FIG. 11d ).

The therapeutic mechanism of action for SIRPabodies is induction ofphagocytosis. Phagocytosis was measured in vitro by coincubating humanmacrophages with Raji-GFP cells in the presence of antibody. Engulfmentof Raji cells by macrophages was detected by flow cytometry. AllSIRPabodies were capable of inducing phagocytosis (FIG. 12).

CD20-2GL-SIRPα was selected as a lead candidate for further studies oftherapeutic efficacy in vivo. A human lymphoma cell line engineered toexpress luciferase was engrafted subcutaneously into NSG mice toestablish a model of localized lymphoma. Treatment with SIRPabodyCD20-2GL-SIRPα resulted in elimination of the lymphoma and increasedsurvival comparable to the synergistic effect seen with co-targetingCD20 and CD47 with combination antibody therapy.

Similar constructs were made for Tlm3 and CD99. The sequence of anexemplary anti-CD99 light chain is provided in SEQ ID NO:12. Thesequence of an exemplary anti-CD99 SIRPabody heavy chain is provided inSEQ ID NO:14, where the signal sequence is amino acid residues 1-19, thelinker sequence is residues 471-480; and the SIRPa binding domain isresidues 481-599.

1. An isolated polypeptide comprising: an immunoglobulin variable regionfused to a sequence comprising a binding domain of SIRPα.
 2. Thepolypeptide of claim 1, comprising: a first and a second polypeptidechain, which first polypeptide chain comprises (i) a first domaincomprising a binding region of a light chain variable domain of animmunoglobulin (V_(L)) specific for a first epitope; and a secondpolypeptide comprising (i) a first domain comprising a binding region ofa heavy chain variable region domain of an immunoglobulin (V_(H))specific for a first epitope; and (ii) a second domain comprising theN-terminal Ig-like domain of SIRPα.
 3. The polypeptide of claim 1,comprising: a first and a second polypeptide chain, which firstpolypeptide chain comprises (i) a first domain comprising a bindingregion of a light chain variable domain of an immunoglobulin (V_(L))specific for a first epitope; and (ii) a second domain comprising theN-terminal Ig-like domain of SIRPα; and a second polypeptide comprising(i) a first domain comprising a binding region of a heavy chain variableregion domain of an immunoglobulin (V_(H)) specific for a first epitope.4. The polypeptide of claim 1, wherein the first polypeptide and thesecond polypeptide further comprise the respective heavy and light chainconstant region domains of the immunoglobulin.
 5. The polypeptide ofclaim 1, wherein the first or the second polypeptide comprises theN-terminal Ig-like domain of SIRPα fused to the amino terminus of theV_(L) or V_(H) domain, respectively.
 6. The polypeptide of claim 1,wherein the first or the second polypeptide comprises the N-terminalIg-like domain of SIRPα fused to the carboxy terminus of the C_(L) orC_(H) domains, respectively.
 7. The polypeptide of claim 1, wherein theSIRPα domain and the V_(H) or C_(H) domains are separated by apolypeptide linker of from 1-20 amino acids in length.
 8. Thepolypeptide of claim 1, comprising the amino acid sequence of SEQ IDNO:1 or a variant thereof.
 9. The polypeptide of claim 1, wherein thevariable domain of the immunoglobulin specifically binds a tumorantigen.
 10. The polypeptide of claim 9, wherein the tumor antigen isCD20.
 11. The polypeptide of claim 9, wherein the tumor antigen is TIM3.12. The polypeptide of claim 9, wherein the tumor antigen is CD99.
 13. Anucleic acid encoding a polypeptide of claim
 1. 14. A pharmaceuticalcomposition comprising the polypeptide of claim
 1. 15. A method oftreating cancer, the method comprising administering an effective doseof a pharmaceutical composition of claim 14 to an individual in needthereof.
 16. The method of claim 15, wherein the cancer is a lymphoma orleukemia.
 17. The method of claim 16, wherein the cancer is aNon-Hodgkin's B cell lymphoma.
 18. The method of claim 16, wherein thecancer is chronic lymphocytic leukemia.
 19. The method of claim 16,wherein the cancer is acute myeloid leukemia.